The present invention relates to parasitic helminth aromatic amino acid decarboxylase (ADC) nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, compounds capable of inhibiting the function of such proteins and methods to identify such inhibitors. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, and/or inhibitors, as well as their use to protect animals from diseases caused by parasitic helminths. The present invention also includes a method for detecting the presence of amino acid decarboxylases.
Parasitic helminth infections in animals, including humans, are typically treated by chemical drugs. One disadvantage with chemical drugs is that they must be administered often. For example, dogs susceptible to heartworm are typically treated monthly. Repeated administration of drugs, however, often leads to the development of resistant helminth strains that no longer respond to treatment. Furthermore, many of the chemical drugs cause harmful side effects in the animals being treated, and as larger doses become required due to the build up of resistance, the side effects become even greater. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection by the parasitic helminth.
An alternative method to prevent parasitic helminth infection includes administering a vaccine against a parasitic helminth. Although many investigators have tried to develop vaccines based on specific antigens, it is well understood that the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting an animal from infection, particularly in the case of parasitic helminths. Although a number of antigens have been identified in several parasitic helminths, including proteases and macromolecules demonstrating protease-like activity, (See, for example, Lustigman, 1995, Antimicrobial Agents and Chemother., 39(9): 1913-1919; Mehta, 1992, Mol. Biochem. Parasitol., 53:1-16; Richer et al., 1992, Exper. Parasitol., 75:213-222; Tripp et al., U.S. Pat. No. 5,569,603) there is yet to be a commercially available vaccine developed for any parasitic helminth.
As an example of the complexity of parasitic helminths, the life cycle of Dirofilaria immitis, the filariid nematode that causes heartworm, includes a variety of life forms, each of which presents different targets, and challenges, for immunization. In a mosquito, D. immitis microfilariae go through two larval stages (L1 and L2) and become mature third stage larvae (L3), which can then be transmitted back to the dog when the mosquito takes a blood meal. In a dog, the L3 molt to the fourth larval stage (L4), and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature to adult heartworms. Adult heartworms are quite large and preferentially inhabit the heart and pulmonary arteries of an animal. Sexually mature adults, after mating, produce microfilariae which traverse capillary beds and circulate in the vascular system of the dog. In particular, heartworm is a major problem in dogs, which typically do not develop immunity upon infection (i.e., dogs can become reinfected even after being cured by chemotherapy). In addition, heartworm infection has been reported in cats, ferrets, and humans.
The mechanisms and regulatory pathways involved in the development of helminths are not clear. For example, it has been shown in the free living nematode, Caenorhabditis elegans (C. elegans), that the development of the larvae is regulated by environmental signals through chemosensory neurons. Blockage of signal transmission affects the development of the nematode (Bargmann, et al., 1991, Science, 251, 1243-1246). Many neuron-related genes have been identified in C. elegans. Mutation of the genes which control normal neuron function in C. elegans will not only affect the behavior of the nematode, but will also affect the development of the larvae and egg laying of mutated female worms. In parasitic nematodes such as D. immitis, very little is known about mechanisms involved in the migration, signal transmission and the developmental regulation of the parasites. However, host and tissue specificities in parasite infections suggest that parasitic nematodes might also need correct environmental signals for development.
There has been no previous report of aromatic amino acid decarboxylases (ADC) in parasitic helminths. Although three genes coding for putative aromatic amino acid decarboxylase-like proteins have been sequenced in the free-living nematode C. elegans, neither biochemical properties nor biological functions of these proteins have been described. An unrelated but biochemically similar molecule, dopa-decarboxylase (DOPA-DC), has been shown to be an important enzyme in catecholamine metabolism in animals. In Drosophila, studies of DOPA-DC indicate that the majority of DOPA-DC is localized in the epidermis and that the enzyme is involved in the formation of flexible cuticle during the development of larvae (see Wright, T. R. F., 1996, Journal of Heredity, 87:175-190). Due to the similarities in biochemical properties and in vivo expression between DOPA-DC and parasitic helminth ADC disclosed herein it is likely that parasitic helminth ADC plays a significant role in cuticle formation during parasitic helminth development and in larval survival in the hostile conditions within the host.
As such, there remains a need to identify efficacious compositions that protect animals against diseases caused by parasitic helminths such as D. immitis. Such compositions would preferably also protect animals from infection by such helminths.
The present invention relates to a novel product and a process to protect animals against parasitic helminth infection (e.g., prevent and/or treat such an infection). According to the present invention there are provided parasitic helminth ADC proteins (e.g. Dirofilaria and Brugia ADC proteins) and mimetopes thereof; parasitic helminth ADC nucleic acid molecules, including those that encode such proteins; antibodies raised against such ADC proteins (i.e., anti-parasitic helminth ADC antibodies); and compounds that inhibit the function of parasitic helminth ADCs (i.e. inhibitory compounds).
The present invention also includes methods to obtain and/or identify such proteins, nucleic acid molecules, antibodies and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitory compounds, as well as use of such therapeutic compositions to protect animals from diseases caused by parasitic helminths.
One embodiment of the present invention is an isolated nucleic acid molecule that includes a parasitic helminth aromatic amino acid decarboxylase nucleic acid molecule. Such nucleic acid molecules are referred to as ADC nucleic acid molecules. A preferred parasitic helminth nucleic acid molecule includes an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule having at least about 50 nucleotides wherein said nucleic acid molecule hybridizes with a nucleic acid molecule selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:21 under conditions that allow about 20% base pair mismatch between said isolated nucleic acid molecule and said nucleic acid molecule having said nucleic acid sequence, and (b) a nucleic acid molecule having at least about 150 nucleotides wherein said nucleic acid sequence hybridizes with a nucleic acid molecule selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:21 under conditions that allow about 30% base pair mismatch between said isolated nucleic acid molecule and said nucleic acid molecule having said nucleic acid sequence.
Additional preferred parasitic helminth nucleic acid molecules include either a Dirofilaria ADC nucleic acid molecule, preferably a Dirofilaria immitis (D. immitis) ADC nucleic acid molecule, or a Brugia ADC nucleic acid molecule, preferably a Brugia malayi (B. malayi) ADC nucleic acid molecule. A D. immitis ADC nucleic acid molecule preferably includes nucleic acid sequence SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16 and/or SEQ ID NO:18, and a B. malayi ADC nucleic acid molecule preferably includes nucleic acid sequence SEQ ID NO:19 and/or SEQ ID NO:21.
In one embodiment, a preferred D. immitis ADC nucleic acid molecule comprises a nucleic acid molecule of at least about 50 nucleotides, preferably at least about 100 nucleotides, more preferably at least about 350 nucleotides, more preferably at least about 450 nucleotides, more preferably at least about 500 nucleotides even more preferably at least about 800 nucleotides. In another embodiment, a preferred B. malayi nucleic acid molecule comprises a nucleic acid molecule of at least about 50 nucleotides, preferably at least about 100 nucleotides, more preferably at least about 350 nucleotides, more preferably at least about 500 nucleotides even more preferably at least about 550 nucleotides. In yet another embodiment, a preferred D. immitis or B. malayi ADC nucleic acid molecule comprises a full-length coding region which encodes a full-length ADC protein.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include an isolated ADC nucleic acid molecule of the present invention. Also included are methods to produce such recombinant molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention includes a parasitic helminth ADC protein. A preferred parasitic helminth ADC protein includes an isolated protein comprising an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:17, and SEQ ID NO:20 or fragments thereof having at least about 35 amino acid residues.
A preferred parasitic helminth ADC protein includes a Dirofilaria or a Brugia ADC protein. More preferred ADC proteins include Dirofilaria immitis or Brugia malayi ADC proteins (referred to herein as Di-ADC or Bm-ADC proteins respectively). A preferred Di-ADC protein comprises amino acid sequence SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:17, and a preferred Bm-ADC protein comprises amino acid sequence SEQ ID NO:20.
In one embodiment, a preferred Di-ADC protein comprises an amino acid sequence of at least about 35 amino acids in length, preferably at least about 50 amino acids in length, more preferably at least about 100 amino acids in length, more preferably at least about 200 amino acids in length, even more preferably at least about 250 amino acids in length. In yet another embodiment, a preferred Bm-ADC protein comprises an amino acid sequence of at least about 35 amino acids in length, preferably at least about 50 amino acids in length, more preferably at least about 100 amino acids in length, more preferably at least about 150 amino acids in length, even more preferably at least about 180 amino acids in length. In yet another embodiment, a preferred Di-ADC or Bm-ADC protein comprises a full-length protein, i.e., a protein encoded by a full-length coding region.
The present invention also relates to mimetopes of parasitic helminth ADC proteins. A preferred mimetope of a parasitic helminth ADC protein includes a mimetope of a Dirofilaria ADC protein or a Brugia ADC protein. The present invention further relates to isolated antibodies that selectively bind to parasitic helminth ADC proteins. A preferred antibody includes an antibody that selectively binds to either a Dirofilaria ADC protein or to a mimetope thereof. Also preferred is an antibody that selectively binds to a Brugia ADC protein or to a mimetope thereof. The present invention further relates to inhibitors of parasitic helminth ADC proteins. A preferred inhibitor of parasitic helminth ADC proteins includes an inhibitor of Dirofilaria ADC protein function or an inhibitor of Brugia ADC protein function. Also included are methods, including recombinant physical or chemical methods, to produce proteins, mimetopes, antibodies, and inhibitors of the present invention. Also included is a method to identify inhibitors of ADC activity which includes the steps of contacting an isolated filariid aromatic amino acid decarboxylase protein having an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:17, and SEQ ID NO:20 with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has aromatic amino acid decarboxylase activity; and determining if said putative inhibitory compound inhibits said activity.
Yet another embodiment of the present invention is a therapeutic composition that is capable of protecting an animal from disease caused by a parasitic helminth. Such a therapeutic composition includes one or more of the following protective compounds: a parasitic helminth ADC protein or a mimetope thereof; an isolated parasitic helminth ADC nucleic acid molecule; an isolated antibody that selectively binds to a parasitic helminth ADC protein; and/or a compound capable of inhibiting ADC function identified by its ability to inhibit parasitic helminth ADC function. A preferred therapeutic composition of the present invention also includes an excipient, an adjuvant and/or a carrier. Preferred ADC nucleic acid molecule therapeutic compositions of the present invention include genetic vaccines, recombinant virus vaccines and recombinant cell vaccines. Also included in the present invention is a method to protect an animal from disease caused by a parasitic helminth, comprising the step of administering to the animal a therapeutic composition of the present invention.
Yet another embodiment of the present invention is a method for detecting the presence of an amino acid decarboxylase, said method comprising the steps of: (a) contacting a putative amino acid decarboxylase-containing composition with a synthetic substrate to create a reaction product, wherein the synthetic substrate comprises an amino acid conjugated to a tag which produces a reference signal, where the tag is cleaved from the synthetic substrate in the presence of an amino acid decarboxylase resulting in a cleavage product which produces a cleavage signal which is different from the reference signal; and (b) observing the reaction signal produced by the reaction product and comparing the reaction signal with the reference signal and the cleavage signal. A reaction signal qualitatively equivalent to the cleavage signal indicates the presence of an amino acid decarboxylase.